N-terminal acetylation (Nt-acetylation) occurs on the majority of eukaryotic proteins and is catalyzed by N-terminal acetyltransferases (NATs). Nt-acetylation is increasingly recognized as a vital modification with functional implications ranging from protein degradation to protein localization. Although early genetic studies in yeast demonstrated that NAT-deletion strains displayed a variety of phenotypes, only recently, the first human genetic disorder caused by a mutation in a NAT gene was reported; boys diagnosed with the X-linked Ogden syndrome harbor a p.Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of the NatA complex, and suffer from global developmental delays and lethality during infancy. Here, we describe a
Saccharomyces cerevisiae model developed by introducing the human wild-type or mutant NatA complex into yeast lacking NatA (NatA-Δ). The wild-type human NatA complex phenotypically complemented the NatA-Δ strain, whereas only a partial rescue was observed for the Ogden mutant NatA complex suggesting that hNaa10 S37P is only partially functional
in vivo. Immunoprecipitation experiments revealed a reduced subunit complexation for the mutant hNatA S37P next to a reduced
in vitro catalytic activity. We performed quantitative Nt-acetylome analyses on a control yeast strain (yNatA), a yeast NatA deletion strain (yNatA-Δ), a yeast NatA deletion strain expressing wild-type human NatA (hNatA), and a yeast NatA deletion strain expressing mutant human NatA (hNatA S37P). Interestingly, a generally reduced degree of Nt-acetylation was observed among a large group of NatA substrates in the yeast expressing mutant hNatA as compared with yeast expressing wild-type hNatA. Combined, these data provide strong support for the functional impairment of hNaa10 S37P
in vivo and suggest that reduced Nt-acetylation of one or more target substrates contributes to the pathogenesis of the Ogden syndrome. Comparative analysis between human and yeast NatA also provided new insights into the co-evolution of the NatA complexes and their substrates. For instance, (Met-)Ala- N termini are more prevalent in the human proteome as compared with the yeast proteome, and hNatA displays a preference toward these N termini as compared with yNatA.Up to 85% of soluble eukaryotic proteins carry an N-terminal acetyl group at their N terminus, which is the result of a co-translational protein modification referred to as N-terminal protein acetylation (Nt-acetylation) or N
α-acetylation (
1). This presumed irreversible protein modification is catalyzed by a specific category of the GCN5-related N-acetyltransferase domain containing superfamily of acetyltransferases; the ribosome associated N-terminal acetyltransferases or NATs
1 (
2). NATs catalyze the acetyl transfer from acetyl coenzyme A (Ac-CoA) to a primary α-amine of the first amino acid residue of a nascent protein chain. In eukaryotes, NATs are composed of at least one catalytic subunit and mainly target different substrate N termini based on their N-terminal sequences (
3).To date, five human NATs hNatA, hNatB, and hNatC; constituting the major human NAT complexes, and hNatD and hNatF have been identified and their substrate specificity characterized (
1,
4–
8). In addition, a putative hNatE complex has been described (
9–
10). Except for NatF, which is only expressed in higher eukaryotes (
1), the substrate specificity profiles of the NatA-E complexes seem to be conserved among eukaryotes (
5–
9,
11–
13).Contrary to the original assumption that Nt-acetylation protected proteins from degradation (
14), it was more recently demonstrated that this modification creates specific degradation signals (termed Ac/N-degrons) in cellular proteins, thereby diversifying this original view substantially. These degrons target at least some Nt-acetylated proteins for the conditional degradation by a novel branch of the N-end rule pathway, an ubiquitin-dependent proteolytic system (
15–
16). In addition, numerous reports implicate Nt-acetylation in cellular differentiation, survival, metabolism, and proliferation, thereby linking it to cancer (
17–
18). As such, Nt-acetylation is now linked to a whole range of molecular implications including protein destabilization and degradation by the Nt-acetylation dependent recruitment of ubiquitin ligases (
15–
16), protein translocation (
19), membrane attachment (
20), and protein complex formation (
21).Among all characterized NATs, NatA displays the broadest substrate specificity profile and thus represents the primary NAT in terms of substrate N termini as it is responsible for the Nt-acetylation of the methionine aminopeptidase (MetAP) iMet-processed serine, threonine, alanine, glycine, and valine starting N termini (
3). The human NatA complex is composed of two essential subunits; the catalytic subunit hNaa10 (hARD1) and the regulatory subunit hNaa15 (NATH/hNAT1) (
4). Deregulations of hNaa10 and/or NatA expression have been linked to various signaling molecules including hypoxia inducible factor-1α, DNA methyltransferase1/E-cadherin, β-catenin/cyclin D1, and Bcl-xL, showing its involvement in hypoxia, tumorigenesis, cell cycle progression, and apoptosis (
17,
22–
26).Recently, the first structures of NATs and a NAT-complex were solved, providing a molecular understanding of the sequence specific Nt-acetylation of protein N termini (
27–
30). Structural analyses of noncomplexed Naa10 and NatA from
Schizosaccharomyces pombe reveal an allosteric modulator function of Naa15 in steering Naa10 specificity and provide a rational for the distinctive substrate specificity profiles observed when assaying non-complexed
versus complexed Naa10 (
10,
27), with both forms co-existing in cells (
10). In particular, three essential catalytic Naa10 residues were found to be incorrectly positioned in non-complexed Naa10, while these shift into the active site in Naa15-complexed Naa10, thereby permitting canonical NatA-mediated Nt-acetylation. Interestingly, noncomplexed Naa10 was shown to efficiently Nt-acetylate glutamate and aspartate starting N termini, whereas poorly acetylating canonical NatA type N termini (
10). The study of Liszczak
et al. further showed that NatA substrate binding specificity was coupled to the catalytic mechanism being used (
27). More specifically, an essential glutamate residue (Glu24 in the protein accession
{"type":"entrez-protein","attrs":{"text":"Q9UTI3","term_id":"74625957","term_text":"Q9UTI3"}}Q9UTI3 (Swiss-Prot)) involved in catalysis, precludes methionine from entering the specificity pocket, whereas cognate NatA substrate N-terminal residues can easily be accommodated. Interestingly, and in contrast to NatA, both wild-type Naa10 and Glu24 mutated Naa10 (Naa10 E24A) were still capable of Nt-acetylating acidic amino acid starting N termini, most likely because of the substrate side-chain carboxyl moiety acting as a functional replacement group in the process of catalysis, whereas essentially no activity could be observed when probing a cognate NatA substrate (
27).Early yeast studies demonstrated that strains with mutated or deleted NAT genes were viable, but displayed a number of different phenotypes (
31). For NatA, the first phenotypes described were defects in sporulation, mating, and entry into stationary phase when
NAA10 (ARD1) was mutated (
32). Four years later, the overlapping phenotypes of
NAA10 and
NAA15 (
NAT1) mutant strains, revealed, along with other data, that Naa10 and Naa15 are in fact components of the NatA acetyltransferase complex (
33–
34). As compared with NatA phenotypes, NatB phenotypes are more severe, including slow growth and defects in mitochondrial inheritance (
35–
36). NatC subunits were initially found to be essential for propagation of the
l-A dsRNA virus, and further for growth on nonfermentable carbon sources (
37–
39). The first reports implicating NAT gene point mutations in human genetic disorders only recently emerged. More specifically, two different point mutations in the X-linked
NAA10 gene were both found to cause developmental delays and were linked to the Ogden syndrome (S37P) (
40) and intellectual disability (R116W) (
41), highlighting the essential importance of NATs and protein Nt-acetylation in biology and disease. Further, in
Caenorhabditis elegans (
42),
Drosophila melanogaster (
43), and
Trypanosoma brucei (
44), Naa10 was proven to be essential and, strengthened by the observed detrimental effects of
NAA10 mutations (
40–
41), the
NAA10 gene function is also believed to be essential in human.Ogden syndrome boys harboring the p.Ser37Pro variant in the gene encoding Naa10 are characterized by craniofacial abnormalities, failure to thrive, developmental delay, hypotonia, cardiac arrhythmias, cryptorchidism, and an aged appearance, ultimately resulting in mortality during infancy (
40). Although this mutation was shown to significantly impair Naa10 catalytic activity
in vitro, we here assessed the influence and functional
in vitro and
in vivo consequences of this mutation on NatA complex formation and NatA activity in a yeast model. By phenotypic screening in yeast, we show that hNaa10 S37P displays a significantly impaired functionality
in vivo. Further, using immunoprecipitation, we show that the human Naa10-Naa15 complex formation is negatively affected by the S37P mutation, and that immunoprecipitated hNatA S37P also displays a reduced
in vitro catalytic activity as compared with wild-type hNatA. Finally, quantitative Nt-acetylome analyses suggest that reduced Nt-acetylation of one or more target substrates contributes to the pathogenesis of the Ogden syndrome.
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